Antisense modulation of caspase 9 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of caspase 9. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding caspase 9. Methods of using these compounds for modulation of caspase 9 expression and for treatment of diseases associated with expression of caspase 9 are provided.

INTRODUCTION

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/659,845 filed Sep. 11, 2000.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods formodulating the expression of caspase 9. In particular, this inventionrelates to compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding caspase 9. Such compounds havebeen shown to modulate the expression of caspase 9.

BACKGROUND OF THE INVENTION

[0003] Apoptosis, or programmed cell death, is a naturally occurringprocess that has been strongly conserved during evolution to preventuncontrolled cell proliferation. This form of cell suicide plays acrucial role in ensuring the development and maintenance ofmulticellular organisms by eliminating superfluous or unwanted cells.However, if this process goes awry becoming overstimulated, cell lossand degenerative disorders including neurological disorders such asAlzheimers, Parkinsons, ALS, retinitis pigmentosa and blood celldisorders can result. Stimuli which can trigger apoptosis include growthfactors such as tumor necrosis factor (TNF), Fas and transforming growthfactor beta (TGFβ), neurotransmitters, growth factor withdrawal, loss ofextracellular matrix attachment and extreme fluctuations inintracellular calcium levels (Afford and Randhawa, Mol. Pathol., 2000,53, 55-63).

[0004] Alternatively, insufficient apoptosis, triggered by growthfactors, extracellular matrix changes, CD40 ligand, viral gene productsneutral amino acids, zinc, estrogen and androgens, can contribute to thedevelopment of cancer, autoimmune disorders and viral infections (Affordand Randhawa, Mol. Pathol., 2000, 53, 55-63). Consequently, apoptosis isregulated under normal circumstances by the interaction of gene productsthat either induce or inhibit cell death and several gene products whichmodulate the apoptotic process have now been identified.

[0005] The most well-characterized apoptotic signaling cascade to dateis that orchestrated by a family of cysteine proteases known ascaspases. These enzymes activate apoptosis through proteolytic eventstriggered by one of several described mechanisms; including ligandbinding to the cell surface death receptors of either the TNF or NGFreceptor families, changes in mitochondrial integrity or chemicalinduction (Thornberry, Br. Med. Bull., 1997, 53, 478-490).

[0006] Caspases have been classified into two groups, initiator caspasesand effector caspases, based upon their position in the apoptoticsignaling pathway.

[0007] Initiator caspases include caspase 1, 2, 4, 5, 8, 9, 10 and 14and these enzymes have the largest prodomains of all the caspasezymogens. These prodomains allow the initiator caspases to interact withother downstream substrates including other caspases. Initiator caspasesare further divided into two groups based on their protein bindingdomains. Caspases 8 and 10 contain the DED (death effector domain) whilecaspases 1, 2, 4 and 9 contain the CARD (caspase recruitment domain)(Bratton et al., Exp. Cell. Res., 2000, 256, 27-33; Garcia-Calvo et al.,Cell. Death Differ., 1999, 6, 362-369).

[0008] Effector caspases are activated by initiator caspases and includecaspase 3, 6, 7, 11 and 13 and these contain a shorter prodomain. Onceactivated, effector caspases then cleave a number of structural andregulatory proteins within the cell (Bratton et al., Exp. Cell. Res.,2000, 256, 27-33; Garcia-Calvo et al., Cell. Death Differ., 1999, 6,362-369).

[0009] Caspase 9 (also known as CASP9, MCH6, APAF3, ICE-LAP6 or ICE9) isa ubiquitously expressed initiator caspase which has been shown to bethe most upstream caspase in the apoptotic cascade and to induceapoptosis in breast carcinoma cells when overexpressed (Duan et al., J.Biol. Chem., 1996, 271, 16720-16724; Kuida, Int. J. Biochem. Cell Biol.,2000, 32, 121-124; Li et al., Cell, 1997, 91, 479-489). Caspase 9 existsas two isoforms in both humans and mice and the shorter of the isoformshas been shown to act as a dominant-negative of the longer form in vitroby blocking protein-protein interactions with the caspase 9 adaptormolecule, Apaf-1 (Fujita et al., Biochem. Biophys. Res. Commun., 1999,264, 550-555; Seol and Billiar, J. Biol. Chem., 1999, 274, 2072-2076;Srinivasula et al., Cancer Res., 1999, 59, 999-1002).

[0010] Activation of the caspase 9 zymogen occurs upon mitochondrialrelease of cytochrome c subsequent to triggers of cell death followed bycomplex formation with Apaf-1 forming the apoptosome. Proteolyticcleavage of the caspase 9 zymogen then results in release of the maturecaspase 9 from the apoptosome and initiates the apoptotic cascade (Salehet al., J. Biol. Chem., 1999, 274, 17941-17945).

[0011] Disclosed in U.S. Pat. No. 6,010,878 are the polypeptide andpolynucleotide sequences of human caspase 9 as well as an expressionvector and host cells for the expression of said vector (Dixit et al.,2000).

[0012] Caspase 9 is required for normal brain development and mediatesapoptosis induced by chemotherapeutic agents (Hakem et al., Cell, 1998,94, 339-352; Kuida, Int. J. Biochem. Cell Biol., 2000, 32, 121-124) andoncogenic transformation (Fearnhead et al., Proc. Natl. Acad. Sci. U. S.A., 1998, 95, 13664-13669; Kuwahara et al., Cancer Lett., 2000, 148,65-71; Soengas et al., Science, 1999, 284, 156-159; Wang et al., Eur. J.Cancer, 1999, 35, 1517-1525; Zhuang and Cohen, Toxicol. Lett., 1998,102-103, 121-129).

[0013] Caspase 9 also plays a role in ischemic recovery (Krajewski etal., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 5752-5757) and thymocyteapoptosis induced by sepsis (Tinsley et al., Shock, 2000, 13, 1-7).These data suggest that modulation of caspase 9 would render opportunityto treat patients with disorders such as cancer, stroke, brain injury orneurodegenerative diseases.

[0014] It is currently believed that modulation of caspase expressionrepresents a potential therapeutic target in a variety of deregulatedapoptotic pathologic conditions. Several types of broad-spectrum caspaseinhbitors have been identified for the treatment of deregulated bonemetabolism (Harada et al., 2000; Reszka, 1999), as immunomodulatoryagents (Gunasekera et al., 2000) and as combination therapies for theregulation of blood cholesterol (Reszka, 1999).

[0015] These non-specific caspase inhibitors fall into three mainclasses; peptide-based molecules that mimic caspase substrates, smallmolecules and naturally-occurring caspase inhibitors or decoys (Deverauxet al., Embo J., 1998, 17, 2215-2223; Dong et al., Biochem. J., 2000,347 Pt 3, 669-677; Gunasekera et al., 2000; Harada et al., 2000; Reszka,1999; Reszka, 1999; Robidoux et al., 2000; Spruce et al., 1999).

[0016] Currently, however, there are no known therapeutic agents whicheffectively inhibit the synthesis of caspase 9 and to date, strategiesaimed at modulating caspase 9 function have involved the use ofantibodies and molecules that block upstream entities such as the deathreceptors, broad-spectrum caspase inhibitors or targeted gene knockoutsin mice. Mice lacking caspase 9 die perinatally with a markedly enlargedand malformed cerebrum caused by reduced apoptosis in the brain andembryonic stem cells lacking caspase 9 show resistance to severalapoptotic stimuli (Hakem et al., Cell, 1998, 94, 339-352; Kuida et al.,Cell, 1998, 94, 325-337).

[0017] There exists, therefore, a need to identify methods of modulatingapoptosis for the therapeutic treatment of human diseases.

[0018] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of gene expression and cellularprocesses.

[0019] The present invention satisfies this need and providescompositions and methods for modulating caspase 9 expression, includingmodulation of aberrant forms of caspase 9, including mutated andalternatively spliced forms.

SUMMARY OF THE INVENTION

[0020] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding caspase 9, and which modulate the expression of caspase 9.Pharmaceutical and other compositions comprising the compounds of theinvention are also provided. Further provided are methods of modulatingthe expression of caspase 9 in cells or tissues comprising contactingsaid cells or tissues with one or more of the antisense compounds orcompositions of the invention. Further provided are methods of treatingan animal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of caspase 9 byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding caspase 9, ultimately modulating theamount of caspase 9 produced. This is accomplished by providingantisense compounds which specifically hybridize with one or morenucleic acids encoding caspase 9. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding caspase 9” encompass DNAencoding caspase 9, RNA (including pre-mRNA and mRNA) transcribed fromsuch DNA, and also cDNA derived from such RNA. The specifichybridization of an oligomeric compound with its target nucleic acidinterferes with the normal function of the nucleic acid. This modulationof function of a target nucleic acid by compounds which specificallyhybridize to it is generally referred to as “antisense”. The functionsof DNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all vital functions suchas, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in or facilitated by the RNA. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of caspase 9. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

[0022] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding caspase 9. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding caspase 9, regardless of the sequence(s) of such codons.

[0023] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0024] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0025] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0026] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0027] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0028] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0029] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0030] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0031] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0032] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0033] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0034] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0035] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0036] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

[0037] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0038] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0039] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0040] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0041] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N— alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0042] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 3′ or 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0043] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0044] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0045] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, which is commonly owned with theinstant application and also herein incorporated by reference.

[0046] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include inter-calators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfadrug, an antidiabetic, an antibacterial or an antibiotic.Oligonucleotide-drug conjugates and their preparation are described inU.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) whichis incorporated herein by reference in its entirety.

[0047] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0048] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds.

[0049] “Chimeric” antisense compounds or “chimeras,” in the context ofthis invention, are antisense compounds, particularly oligonucleotides,which contain two or more chemically distinct regions, each made up ofat least one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0050] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0051] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0052] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0053] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0054] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0055] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0056] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0057] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0058] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0059] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of caspase 9 is treated by administering antisense compoundsin accordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0060] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding caspase 9, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding caspase 9can be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of caspase 9 in a sample mayalso be prepared.

[0061] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0062] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0063] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of whichis incorporated herein by reference in their entirety.

[0064] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0065] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0066] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0067] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0068] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0069] Emulsions

[0070] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0071] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0072] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0073] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0074] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0075] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0076] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0077] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0078] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0079] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0080] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C₈-C₁₀glycerides, vegetable oils and silicone oil.

[0081] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0082] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0083] Liposomes

[0084] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0085] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0086] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0087] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0088] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0089] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0090] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0091] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0092] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0093] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0094] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0095] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0096] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765). Various liposomes comprising one or more glycolipids areknown in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987,507, 64) reported the ability of monosialoganglioside G_(M1),galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G_(M1) or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0097] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0098] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0099] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0100] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0101] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0102] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0103] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0104] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0105] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0106] Penetration Enhancers

[0107] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0108] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0109] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0110] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0111] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0112] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0113] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0114] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0115] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0116] Carriers

[0117] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0118] Excipients

[0119] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0120] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0121] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0122] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0123] Other Components

[0124] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0125] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0126] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0127] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0128] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0129] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0130] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy Amidites

[0131] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0132] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

[0133] 2′-Fluoro Amidites

[0134] 2′-Fluorodeoxyadenosine Amidites

[0135] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0136] 2′-Fluorodeoxyguanosine

[0137] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0138] 2′-Fluorouridine

[0139] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0140] 2′-Fluorodeoxycytidine

[0141] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0142] 2′-O-(2-Methoxyethyl) Modified Amidites

[0143] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0144] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0145] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

[0146] 2′-O-Methoxyethyl-5-methyluridine

[0147] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0148] 2′-O-Methoxyethyl-51-O-dimethoxytrityl-5-methyluridine

[0149] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0150]3′-O-Acetyl-21-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0151] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0152]31-O-Acetyl-21-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0153] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0154] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0155] A solution of3¹-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0156]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0157] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0158]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0159]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0160] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl)nucleoside Amidites

[0161] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0162] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0163] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0164] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0165]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0166] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0167]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0168]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅, under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0169]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0170]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂c1₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

[0171] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0172] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0173] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0174] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0175]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0176] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂OS under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0177] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0178] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0179]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0180] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 Al 940203.)Standard protection procedures should afford2¹-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0181] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0182] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0183] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0184] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0185]5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methylUridine

[0186] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂,Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

[0187]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0188] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

[0189] Oligonucleotide Synthesis

[0190] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0191] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0192] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0193] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0194] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0195] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0196] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0197] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0198] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3 Oligonucleoside Synthesis

[0199] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0200] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0201] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0202] PNA Synthesis

[0203] Peptide nucleic acids (PNAS) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0204] Synthesis of Chimeric Oligonucleotides

[0205] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0206] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0207] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[0208] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0209] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[0210] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0211] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0212] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0213] Oligonucleotide Isolation

[0214] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0215] Oligonucleotide Synthesis—96 Well Plate Format

[0216] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0217] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

[0218] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0219] Cell Culture and Oligonucleotide Treatment

[0220] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 5 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0221] T-24 Cells:

[0222] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0223] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0224] A549 Cells:

[0225] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0226] NHDF Cells:

[0227] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0228] HEK cells:

[0229] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville Md.). HEKs were routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

[0230] b.END Cells:

[0231] The mouse brain endothelial cell line b.END was obtained from Dr.Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.ENDcells were routinely cultured in DMEM, high glucose (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 3000 cells/well for use in RT-PCR analysis.

[0232] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0233] Treatment with Antisense Compounds:

[0234] When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0235] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0236] Analysis of Oligonucleotide Inhibition of Caspase 9 Expression

[0237] Antisense modulation of caspase 9 expression can be assayed in avariety of ways known in the art. For example, caspase 9 mRNA levels canbe quantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0238] Protein levels of caspase 9 can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to caspase 9 can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0239] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0240] Poly(A)+ mRNA Isolation

[0241] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 mL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0242] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0243] Total RNA Isolation

[0244] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 100 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 100 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPEwas then added to each well of the RNEASY 96™ plate and the vacuumapplied for a period of 15 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 10 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 60 μL water into each well, incubating1 minute, and then applying the vacuum for 30 seconds. The elution stepwas repeated with an additional 60 μL water.

[0245] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0246] Real-Time Quantitative PCR Analysis of Caspase 9 mRNA Levels

[0247] Quantitation of caspase 9 mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0248] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0249] PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μL PCRcocktail (1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0250] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,Analytical Biochemistry, 1998, 265, 368-374.

[0251] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 25 uL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0252] Probes and primers to human caspase 9 were designed to hybridizeto a human caspase 9 sequence, using published sequence information(GenBank accession number U60521, incorporated herein as SEQ ID NO:3).For human caspase 9 the PCR primers were:

[0253] forward primer: ATTGTGGGATGTTCAGCACTGT (SEQ ID NO: 4)

[0254] reverse primer: TGTTTGGCACCACTCAGGAA (SEQ ID NO: 5) and the PCRprobe was: FAM-CCTTGCCTCAATGCCAGTAACGCG-TAMRA

[0255] (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City,Calif.) is the fluorescent reporter dye) and TAMRA (PE-AppliedBiosystems, Foster City, Calif.) is the quencher dye. For human GAPDHthe PCR primers were:

[0256] forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)

[0257] reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8) and thePCR probe was: 5′ JOE-CGCCTGGTCACCAGGGCTGCT-TAMPA 3′ (SEQ ID NO: 9)where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

[0258] Probes and primers to mouse caspase 9 were designed to hybridizeto a mouse caspase 9 sequence, using published sequence information(GenBank accession number AB019600, incorporated herein as SEQ IDNO:10). For mouse caspase 9 the PCR primers were:

[0259] forward primer: CCAACTTGGACCGTGACAAA (SEQ ID NO:11)

[0260] reverse primer: CGTTCTTCACCTCCACCATGA (SEQ ID NO: 12) and the PCRprobe was: FAM-TTGAGCACCGATTCCGCTGGCT-TAMRA (SEQ ID NO: 13) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For mouse GAPDH the PCR primers were:

[0261] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)

[0262] reverse primer: GGGTCTCGCTCCTGGAAGCT (SEQ ID NO: 15) and the PCRprobe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16)where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14

[0263] Northern Blot Analysis of Caspase 9 mRNA Levels

[0264] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0265] To detect human caspase 9, a human caspase 9 specific probe wasprepared by PCR using the forward primer ATTGTGGGATGTTCAGCACTGT (SEQ IDNO: 4) and the reverse primer TGTTTGGCACCACTCAGGAA (SEQ ID NO: 5). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0266] To detect mouse caspase 9, a mouse caspase 9 specific probe wasprepared by PCR using the forward primer CCAACTTGGACCGTGACAAA (SEQ IDNO:11) and the reverse primer CGTTCTTCACCTCCACCATGA (SEQ ID NO: 12). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0267] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0268] Antisense Inhibition of Human Caspase 9 Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0269] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humancaspase 9 RNA, using published sequences (GenBank accession numberU60521, incorporated herein as SEQ ID NO: 3, GenBank accession numberAL046716, incorporated herein as SEQ ID NO: 17, GenBank accession numberAB015653, incorporated herein as SEQ ID NO: 18, GenBank accession numberAB019198, incorporated herein as SEQ ID NO: 19, GenBank accession numberAB019199, incorporated herein as SEQ ID NO: 20, GenBank accession numberAB019200, incorporated herein as SEQ ID NO: 21, GenBank accession numberAB019201, incorporated herein as SEQ ID NO: 22, GenBank accession numberAB019202, incorporated herein as SEQ ID NO: 23, GenBank accession numberAB019203, incorporated herein as SEQ ID NO: 24, and GenBank accessionnumber AB019204, incorporated herein as SEQ ID NO: 25). Theoligonucleotides are shown in Table 1.“Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humancaspase 9 mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from two experiments. Ifpresent, “N.D.” indicates “no data”. TABLE 1 Inhibition of human caspase9 mRNA levels by chimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ IDNO SITE SEQUENCE % INHIB NO 135222 5′ UTR 3 21 gtagccaactaagactccag 7426 135223 Start 3 37 gcttcgtccatggcgagtag 79 27 Codon 135224 Coding 3169 gagcctgcccgctggatgtc 96 28 135225 Coding 3 193 ctggcctgatcccgccgaga63 29 135226 Coding 3 199 agctgcctggcctgatcccg 78 30 135227 Coding 3 208tctatgatcagctgcctggc 78 31 135228 Coding 3 264 tgtcctctaagcaggagatg 9532 135229 Coding 3 270 ggcctgtgtcctctaagcag 85 33 135230 Coding 3 283gccagcatgtcctggcctgt 91 34 135231 Coding 3 308 ttgcctgttagttcgcagaa 9135 135232 Coding 3 391 ctgagaacctctggtttgcg 96 36 135233 Coding 3 442acatcaccaaatcctccaga 36 37 135234 Coding 3 455 ctcaagagcaccgacatcac 5938 135235 Coding 3 472 gcatttcccctcaaactctc 88 39 135236 Coding 3 490aggatgtaagccaaatctgc 51 40 135237 Coding 3 500 ctccatgctcaggatgtaag 9441 135238 Coding 3 520 atgaggcagtggccacaggg 43 42 135239 Coding 3 541cagaagttcacattgttgat 78 43 135240 Coding 3 599 ccgcaacttctcacagtcga 9044 135241 Coding 3 652 gcagtcaggtcgcccttcac 76 45 135242 Coding 3 658ttcttggcagtcaggtcgcc 81 46 135243 Coding 3 662 cattttcttggcagtcaggt 9047 135244 Coding 3 666 gcaccattttcttggcagtc 62 48 135245 Coding 3 707cagagcaccgtggtcctgct 83 49 135246 Coding 3 724 accaccacgcagcagtccag 8450 135247 Coding 3 745 tgacagccgtgagagagaat 79 51 135248 Coding 3 753ggctggcctgacagccgtga 76 52 135249 Coding 3 756 ggtggctggcctgacagccg 4453 135250 Coding 3 782 gccgtagacagcccctggga 77 54 135251 Coding 3 791tccatctgtgccgtagacag 86 55 135252 Coding 3 836 ggtcccattgaagatgttca 6156 135253 Coding 3 884 ggcctggatgaaaaagagct 52 57 135254 Coding 3 1158cagagtgagcccactgctca 61 58 135255 Coding 3 1174 agggactgcaggtcttcaga 2959 135256 Coding 3 1194 cattagcgaccctaagcagg 87 60 135257 Coding 3 1228ggcatctgtttataaatccc 93 61 135258 Stop 3 1286 gccctggccttatgatgttt 64 62Codon 135259 3′ UTR 3 1358 aagtccaggcctcagcctct 82 63 135260 3′ UTR 31362 aggaaagtccaggcctcagc 75 64 135261 3′ UTR 3 1386ccggctgcaaagtccttgag 93 65 135262 3′ UTR 3 1394 accctgtgccggctgcaaag 8266 135263 3′ UTR 3 1438 ggaagctgctaagagcctgt 87 67 135264 3′ UTR 3 1468cctccactgttcagcacttg 96 68 135265 3′ UTR 3 1484 ttcatctgtccctcttcctc 4069 135266 3′ UTR 3 1503 ccacgtgcaatccacggcat 95 70 135267 3′ UTR 3 1513gctcaagaggccacgtgcaa 83 71 135268 3′ UTR 3 1552 gatcatgggacacaagtcac 8172 135269 3′ UTR 3 1692 aggaagacgcgttactggca 97 73 135270 3′ UTR 3 1715aacctttttgtttggcacca 95 74 135271 3′ UTR 3 1828 gtttgctgatctatgagcga 8475 135272 3′ UTR 3 1831 ccggtttgctgatctatgag 79 76 135273 3′ UTR 3 1943catatttgcctcagatttat 84 77 135274 3′ UTR 3 1944 ccatatttgcctcagattta 8578 135275 Exon 17 183 ggacgcatctccaaggcctc 1 79 135276 Exon 17 189gcctccggacgcatctccaa 60 80 135277 Exon 17 210 ggtcagtcttcgctccccac 45 81135278 Exon 17 215 ccccgggtcagtcttcgctc 31 82 135279 Exon 17 219acggccccgggtcagtcttc 48 83 135280 Coding 18 429 ctttctgctcgacatcacca 084 135281 Intron 19 847 ctatctcaaaggcaggaaaa 74 85 135282 Intron 20 361tcaagagcacctgaagaggc 91 86 135283 Exon 20 396 ggagactcaccaaatctgca 89 87135284 Intron 20 425 agagcccgagctactggccc 81 88 135285 Intron 21 349acagggacccacgtaaaccc 73 89 135286 Intron 21 367 ggatgtaagcctgccagcac 7590 135287 Intron 21 544 gtgagtgtaccttggcagtc 82 91 135288 Intron 21 728gccgagagagccagtcctca 81 92 135289 Intron 22 252 gcaccattttctacaagaga 8193 135290 Exon 22 342 ggcttcctacctgacagccg 73 94 135291 Intron 23 232ctgttcagaggttttgtggg 57 95 135292 Intron 23 250 gagtaggaatccaacagcct 8696 135293 Intron 23 300 ggtggctggcctagaagacc 60 97 135294 Exon 23 450ggagctgcttacccccacca 89 98 135295 Intron 24 253 acctcttgtcattgaaatac 3299 135296 Intron 24 284 tctttctgctctgcaggaag 63 100 135297 Intron 25 196gaccttgactcatacataca 85 101 135298 Intron 25 464 aggaggacacgctgcccctc 80102 135299 Intron 25 688 gcactgaggaccagtccatc 63 103

[0270] As shown in Table 1, SEQ ID NOs 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72,73, 74, 75, 76, 77, 78, 80, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 100, 101, 102 and 103 demonstrated at least 40%inhibition of human caspase 9 expression in this assay and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “active sites” and are thereforepreferred sites for targeting by compounds of the present invention.

Example 16

[0271] Antisense Inhibition of Mouse Caspase 9 Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap.

[0272] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mousecaspase 9 RNA, using published sequences (GenBank accession numberAB019600, incorporated herein as SEQ ID NO: 10, and GenBank accessionnumber AB019601, incorporated herein as SEQ ID NO: 104). Theoligonucleotides are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mousecaspase 9 mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from two experiments. Ifpresent, “N.D.” indicates “no data”. TABLE 2 Inhibition of mouse caspase9 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NOSITE SEQUENCE % INHIB NO 135225 Coding 10 148 ctggcctgatcccgccgaga 87 29135226 Coding 10 154 agctgcctggcctgatcccg 0 30 135228 Coding 10 219tgtcctctaagcaggagatg 70 32 135229 Coding 10 225 ggcctgtgtcctctaagcag 1033 135238 Coding 10 589 atgaggcagtggccacaggg 75 42 135239 Coding 10 610cagaagttcacattgttgat 33 43 135243 Coding 10 731 cattttcttggcagtcaggt 5447 135255 Coding 10 1243 agggactgcaggtcttcaga 0 59 135309 Coding 10 22accctgcatcgccgcaggag 56 105 135310 Coding 10 24 gcaccctgcatcgccgcagg 33106 135311 Coding 10 32 cactaggcgcaccctgcatc 24 107 135312 Coding 10 112tgaatatcctcgatcatgtc 63 108 135313 Coding 10 113 ctgaatatcctcgatcatgt 79109 135314 Coding 10 117 cctgctgaatatcctcgatc 52 110 135315 Coding 10136 cgccgagacccagatcctgc 86 111 135316 Coding 10 168caaggtctgtgaccagctgc 69 112 135317 Coding 10 171 tctcaaggtctgtgaccagc 71113 135318 Coding 10 181 ctccctcgggtctcaaggtc 55 114 135319 Coding 10200 gaagagaggaagggcctgcc 7 115 135320 Coding 10 240 aagccagggtgccttggcct86 116 135321 Coding 10 251 ttgcaagagtgaagccaggg 84 117 135322 Coding 10262 tgccgaccgctttgcaagag 73 118 135323 Coding 10 314aggcaccaggtggtctaggg 17 119 135324 Coding 10 350 ctcctttgctgtgagtccca 29120 135325 Coding 10 353 ctgctcctttgctgtgagtc 0 121 135326 Coding 10 375acggctccagcttcactact 47 122 135327 Coding 10 379 tgtgacggctccagcttcac 22123 135328 Coding 10 383 aggctgtgacggctccagct 70 124 135329 Coding 10388 acggcaggctgtgacggctc 36 125 135330 Coding 10 395gtttcccacggcaggctgtg 68 126 135331 Coding 10 459 tgagaacctctggcttgagc 74127 135332 Coding 10 461 tctgagaacctctggcttga 49 128 135333 Coding 10464 tggtctgagaacctctggct 84 129 135334 Coding 10 490ccaatgtccaccggcctggg 13 130 135335 Coding 10 534 ccctgatcttccctggaaca 70131 135336 Coding 10 549 ccatatctgcatgtcccctg 32 132 135337 Coding 10560 cagggtgtatgccatatctg 33 133 135338 Coding 10 564aatccagggtgtatgccata 49 134 135339 Coding 10 566 cgaatccagggtgtatgcca 71135 135340 Coding 10 599 attgttgatgatgaggcagt 68 136 135341 Coding 10601 acattgttgatgatgaggca 78 137 135342 Coding 10 659gtcacggtccaagttggagc 7 138 135343 Coding 10 670 tgctcaagtttgtcacggtc 84139 135344 Coding 10 681 agcggaatcggtgctcaagt 89 140 135345 Coding 10707 cttcacctccaccatgaagc 41 141 135346 Coding 10 708tcttcacctccaccatgaag 10 142 135347 Coding 10 726 tcttggcagtcaggtcgttc 69143 135348 Coding 10 772 gcacggtggttccggtgtgc 61 144 135349 Coding 10783 agcagtccagggcacggtgg 81 145 135350 Coding 10 789ccacaaagcagtccagggca 69 146 135351 Coding 10 800 gaggatgaccaccacaaagc 44147 135352 Coding 10 817 gcctggcagccatgagagag 10 148 135353 Coding 10824 gtggctggcctggcagccat 0 149 135354 Coding 10 845 gacagcacccgggaactgga62 150 135355 Coding 10 890 attcacaattttctcaatgg 6 151 135356 Coding 10984 caaagccatggtctttctgc 61 152 135357 Coding 10 993aggccacctcaaagccatgg 3 153 135358 Coding 10 1015 gtcctgccttgagaggaagt 66154 135359 Coding 10 1040 atctggctcagagtcactgt 82 155 135360 Coding 101087 gcatccagctggtccaaggg 39 156 135361 Coding 10 1095ttgacacagcatccagctgg 71 157 135362 Coding 10 1104 taggcaaacttgacacagca72 158 135363 Coding 10 1106 ggtaggcaaacttgacacag 76 159 135364 Coding10 1137 aggtggagtaggacacaagg 27 160 135365 Coding 10 1146aacctgggaaggtggagtag 45 161 135366 Coding 10 1169 tttcttgtccctccaggaga62 162 135367 Coding 10 1177 gagccacttttcttgtccct 82 163 135368 Coding10 1183 taccaggagccacttttctt 66 164 135369 Coding 10 1187gatgtaccaggagccacttt 67 165 135370 Coding 10 1198 tccaaggtctcgatgtacca70 166 135371 Coding 10 1207 agaatgccatccaaggtctc 23 167 135372 Coding10 1215 actgctccagaatgccatcc 59 168 135373 Coding 10 1255accctgagaaggagggactg 66 169 135374 Coding 10 1284 aagtccctttctcagaaaca44 170 135375 Coding 10 1303 cagccaggaatctgcttgta 90 171 135376 Coding10 1323 ttttccggaggaagttaaaa 77 172 135377 Coding 10 1328cagctttttccggaggaagt 93 173 135378 Coding 104 1154 gcattggcgacctgggaagg53 174

[0273] As shown in Table 2, SEQ ID NOs 29, 32, 42, 43, 47, 105, 106,108, 109, 110, 111, 112, 113, 114, 116, 117, 118, 122, 124, 125, 126,127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 143,144, 145, 146, 147, 150, 152, 154, 155, 156, 157, 158, 159, 161, 162,163, 164, 165, 166, 168, 169, 170, 171, 172, 173 and 174 demonstrated atleast 30% inhibition of mouse caspase 9 expression in this experimentand are therefore preferred. The target sites to which these preferredsequences are complementary are herein referred to as “active sites” andare therefore preferred sites for targeting by compounds of the presentinvention.

Example 17

[0274] Western Blot Analysis of Caspase 9 Protein Levels

[0275] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to caspase 9 is used,with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 174 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 atgcattctg cccccaagga 20 3 1963 DNA Homo sapiens CDS(46)...(1296) 3 cggaagcgga ctgaggcggc ctggagtctt agttggctac tcgcc atggac gaa gcg 57 Met Asp Glu Ala 1 gat cgg cgg ctc ctg cgg cgg tgc cgg ctgcgg ctg gtg gaa gag ctg 105 Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu ArgLeu Val Glu Glu Leu 5 10 15 20 cag gtg gac cag ctc tgg gac gcc ctg ctgagc agc gag ctg ttc agg 153 Gln Val Asp Gln Leu Trp Asp Ala Leu Leu SerSer Glu Leu Phe Arg 25 30 35 ccc cat atg atc gag gac atc cag cgg gca ggctct gga tct cgg cgg 201 Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly SerGly Ser Arg Arg 40 45 50 gat cag gcc agg cag ctg atc ata gat ctg gag actcga ggg agt cag 249 Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr ArgGly Ser Gln 55 60 65 gct ctt cct ttg ttc atc tcc tgc tta gag gac aca ggccag gac atg 297 Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr Gly GlnAsp Met 70 75 80 ctg gct tcg ttt ctg cga act aac agg caa gca gca aag ttgtcg aag 345 Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala Lys Leu SerLys 85 90 95 100 cca acc cta gaa aac ctt acc cca gtg gtg ctc aga cca gagatt cgc 393 Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg Pro Glu IleArg 105 110 115 aaa cca gag gtt ctc aga ccg gaa aca ccc aga cca gtg gacatt ggt 441 Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro Val Asp IleGly 120 125 130 tct gga gga ttt ggt gat gtc ggt gct ctt gag agt ttg agggga aat 489 Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser Leu Arg GlyAsn 135 140 145 gca gat ttg gct tac atc ctg agc atg gag ccc tgt ggc cactgc ctc 537 Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His CysLeu 150 155 160 att atc aac aat gtg aac ttc tgc cgt gag tcc ggg ctc cgcacc cgc 585 Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg ThrArg 165 170 175 180 act ggc tcc aac atc gac tgt gag aag ttg cgg cgt cgcttc tcc tcg 633 Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg PheSer Ser 185 190 195 ccg cat ttc atg gtg gag gtg aag ggc gac ctg act gccaag aaa atg 681 Pro His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala LysLys Met 200 205 210 gtg ctg gct ttg ctg gag ctg gcg cag cag gac cac ggtgct ctg gac 729 Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His Gly AlaLeu Asp 215 220 225 tgc tgc gtg gtg gtc att ctc tct cac ggc tgt cag gccagc cac ctg 777 Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln Ala SerHis Leu 230 235 240 cag ttc cca ggg gct gtc tac ggc aca gat gga tgc cctgtg tcg gtc 825 Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro ValSer Val 245 250 255 260 gag aag att gtg aac atc ttc aat ggg acc agc tgcccc agc ctg gga 873 Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys ProSer Leu Gly 265 270 275 gga aag ccc aag ctc ttt ttc atc cag gcc tgt ggtggg gag cag aaa 921 Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly GlyGlu Gln Lys 280 285 290 gac cat ggg ttt gag gtg gcc tcc act tcc cct gaagac gag tcc cct 969 Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu AspGlu Ser Pro 295 300 305 ggc agt aac ccc gag cca gat gcc acc ccg ttc caggaa ggt ttg agg 1017 Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln GluGly Leu Arg 310 315 320 acc ttc gac cag ctg gac gcc ata tct agt ttg cccaca ccc agt gac 1065 Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro ThrPro Ser Asp 325 330 335 340 atc ttt gtg tcc tac tct act ttc cca ggt tttgtt tcc tgg agg gac 1113 Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe ValSer Trp Arg Asp 345 350 355 ccc aag agt ggc tcc tgg tac gtt gag acc ctggac gac atc ttt gag 1161 Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu AspAsp Ile Phe Glu 360 365 370 cag tgg gct cac tct gaa gac ctg cag tcc ctcctg ctt agg gtc gct 1209 Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu LeuLeu Arg Val Ala 375 380 385 aat gct gtt tcg gtg aaa ggg att tat aaa cagatg cct ggt tgc ttt 1257 Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln MetPro Gly Cys Phe 390 395 400 aat ttc ctc cgg aaa aaa ctt ttc ttt aaa acatca taa ggccagggcc 1306 Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser405 410 415 cctcaccctg ccttatcttg caccccaaag ctttcctgcc ccaggcctgaaagaggctga 1366 ggcctggact ttcctgcaac tcaaggactt tgcagccggc acagggtctgctctttctct 1426 gccagtgaca gacaggctct tagcagcttc cagattgacg acaagtgctgaacagtggag 1486 gaagagggac agatgaatgc cgtggattgc acgtggcctc ttgagcagtggctggtccag 1546 ggctagtgac ttgtgtccca tgatccctgt gttgtctcta gagcagggattaacctctgc 1606 actactgaca tgtggggcca ggtcaccctt tgctgtgagg ctgtcctgtacattgtggga 1666 tgttcagcac tgtcccttgc ctcaatgcca gtaacgcgtc ttcctgagtggtgccaaaca 1726 aaaaggttct caggtgttgc caaatatgtc ctggggtata aaactttcctcgcctgacaa 1786 ccactggtct gtagggattt ttggctacac acaaaccagt atcgctcatagatcagcaaa 1846 ccggggccta ctagagtctg aacagctgta atctatgaat tctaagtgaaattttaaaaa 1906 ttgttaattt ttcctatatt gcattaattt taaaaaataa atctgaggcaaatatgg 1963 4 22 DNA Artificial Sequence PCR Primer 4 attgtgggatgttcagcact gt 22 5 20 DNA Artificial Sequence PCR Primer 5 tgtttggcaccactcaggaa 20 6 24 DNA Artificial Sequence PCR Probe 6 ccttgcctcaatgccagtaa cgcg 24 7 21 DNA Artificial Sequence PCR Primer 7 caacggatttggtcgtattg g 21 8 26 DNA Artificial Sequence PCR Primer 8 ggcaacaatatccactttac cagagt 26 9 21 DNA Artificial Sequence PCR Probe 9 cgcctggtcaccagggctgc t 21 10 1365 DNA Mus musculus CDS (1)...(1365) 10 atg gac gaggcg gac cgg cag ctc ctg cgg cga tgc agg gtg cgc cta 48 Met Asp Glu AlaAsp Arg Gln Leu Leu Arg Arg Cys Arg Val Arg Leu 1 5 10 15 gtg agc gagctg caa gtc gcg gag ctc tgg gac gct ctg ctg agt cga 96 Val Ser Glu LeuGln Val Ala Glu Leu Trp Asp Ala Leu Leu Ser Arg 20 25 30 gag ctc ttc acgcgc gac atg atc gag gat att cag cag gca gga tct 144 Glu Leu Phe Thr ArgAsp Met Ile Glu Asp Ile Gln Gln Ala Gly Ser 35 40 45 ggg tct cgg cgg gatcag gcc agg cag ctg gtc aca gac ctt gag acc 192 Gly Ser Arg Arg Asp GlnAla Arg Gln Leu Val Thr Asp Leu Glu Thr 50 55 60 cga ggg agg cag gcc cttcct ctc ttc atc tcc tgc tta gag gac aca 240 Arg Gly Arg Gln Ala Leu ProLeu Phe Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 ggc caa ggc acc ctg gcttca ctc ttg caa agc ggt cgg caa gca gcc 288 Gly Gln Gly Thr Leu Ala SerLeu Leu Gln Ser Gly Arg Gln Ala Ala 85 90 95 aag cag gat cca gag gct gttaaa ccc cta gac cac ctg gtg cct gtg 336 Lys Gln Asp Pro Glu Ala Val LysPro Leu Asp His Leu Val Pro Val 100 105 110 gtc ctg gga cca atg gga ctcaca gca aag gag cag aga gta gtg aag 384 Val Leu Gly Pro Met Gly Leu ThrAla Lys Glu Gln Arg Val Val Lys 115 120 125 ctg gag ccg tca cag cct gccgtg gga aac ctc acc cca gtg gtg ctg 432 Leu Glu Pro Ser Gln Pro Ala ValGly Asn Leu Thr Pro Val Val Leu 130 135 140 ggg cca gaa gag ctc tgg cctgct cgg ctc aag cca gag gtt ctc aga 480 Gly Pro Glu Glu Leu Trp Pro AlaArg Leu Lys Pro Glu Val Leu Arg 145 150 155 160 cca gaa aca ccc agg ccggtg gac att ggt tct ggc gga gct cat gat 528 Pro Glu Thr Pro Arg Pro ValAsp Ile Gly Ser Gly Gly Ala His Asp 165 170 175 gtc tgt gtt cca ggg aagatc agg gga cat gca gat atg gca tac acc 576 Val Cys Val Pro Gly Lys IleArg Gly His Ala Asp Met Ala Tyr Thr 180 185 190 ctg gat tcg gat ccc tgtggc cac tgc ctc atc atc aac aat gtg aac 624 Leu Asp Ser Asp Pro Cys GlyHis Cys Leu Ile Ile Asn Asn Val Asn 195 200 205 ttc tgc cct tcc tcg gggctc ggc aca cgc acg ggc tcc aac ttg gac 672 Phe Cys Pro Ser Ser Gly LeuGly Thr Arg Thr Gly Ser Asn Leu Asp 210 215 220 cgt gac aaa ctt gag caccga ttc cgc tgg ctg cgc ttc atg gtg gag 720 Arg Asp Lys Leu Glu His ArgPhe Arg Trp Leu Arg Phe Met Val Glu 225 230 235 240 gtg aag aac gac ctgact gcc aag aaa atg gtc acg gct ttg atg gag 768 Val Lys Asn Asp Leu ThrAla Lys Lys Met Val Thr Ala Leu Met Glu 245 250 255 atg gca cac cgg aaccac cgt gcc ctg gac tgc ttt gtg gtg gtc atc 816 Met Ala His Arg Asn HisArg Ala Leu Asp Cys Phe Val Val Val Ile 260 265 270 ctc tct cat ggc tgccag gcc agc cac ctc cag ttc ccg ggt gct gtc 864 Leu Ser His Gly Cys GlnAla Ser His Leu Gln Phe Pro Gly Ala Val 275 280 285 tat ggg aca gat ggatgc tcc gtg tcc att gag aaa att gtg aat atc 912 Tyr Gly Thr Asp Gly CysSer Val Ser Ile Glu Lys Ile Val Asn Ile 290 295 300 ttc aac ggg agc ggctgc ccc agc ctg gga ggg aag ccc aag ctc ttc 960 Phe Asn Gly Ser Gly CysPro Ser Leu Gly Gly Lys Pro Lys Leu Phe 305 310 315 320 ttc atc cag gcctgc ggt ggt gag cag aaa gac cat ggc ttt gag gtg 1008 Phe Ile Gln Ala CysGly Gly Glu Gln Lys Asp His Gly Phe Glu Val 325 330 335 gcc tgc act tcctct caa ggc agg acc ttg gac agt gac tct gag cca 1056 Ala Cys Thr Ser SerGln Gly Arg Thr Leu Asp Ser Asp Ser Glu Pro 340 345 350 gat gct gtc ccatat cag gaa ggc cca agg ccc ttg gac cag ctg gat 1104 Asp Ala Val Pro TyrGln Glu Gly Pro Arg Pro Leu Asp Gln Leu Asp 355 360 365 gct gtg tca agtttg cct acc ccc agt gac atc ctt gtg tcc tac tcc 1152 Ala Val Ser Ser LeuPro Thr Pro Ser Asp Ile Leu Val Ser Tyr Ser 370 375 380 acc ttc cca ggtttt gtc tcc tgg agg gac aag aaa agt ggc tcc tgg 1200 Thr Phe Pro Gly PheVal Ser Trp Arg Asp Lys Lys Ser Gly Ser Trp 385 390 395 400 tac atc gagacc ttg gat ggc att ctg gag cag tgg gct cgc tct gaa 1248 Tyr Ile Glu ThrLeu Asp Gly Ile Leu Glu Gln Trp Ala Arg Ser Glu 405 410 415 gac ctg cagtcc ctc ctt ctc agg gtc gcc aat gct gtt tct gag aaa 1296 Asp Leu Gln SerLeu Leu Leu Arg Val Ala Asn Ala Val Ser Glu Lys 420 425 430 ggg act tacaag cag att cct ggc tgt ttt aac ttc ctc cgg aaa aag 1344 Gly Thr Tyr LysGln Ile Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys 435 440 445 ctg ttt tttaaa act tca tga 1365 Leu Phe Phe Lys Thr Ser 450 11 20 DNA ArtificialSequence PCR Primer 11 ccaacttgga ccgtgacaaa 20 12 21 DNA ArtificialSequence PCR Primer 12 cgttcttcac ctccaccatg a 21 13 22 DNA ArtificialSequence PCR Probe 13 ttgagcaccg attccgctgg ct 22 14 20 DNA ArtificialSequence PCR Primer 14 ggcaaattca acggcacagt 20 15 20 DNA ArtificialSequence PCR Primer 15 gggtctcgct cctggaagct 20 16 27 DNA ArtificialSequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 27 17 603 DNA Homosapiens 17 gacgcagtgg tggattctgg agcggggcgg tgacgtgggg cgggttctggggcggggcgg 60 tgacgcgagc gtgttctggg gcggggcggt gatgggaggc ttggccctgggggcggggcg 120 aggcgcagag gtgcgtcctg agggcggggc ggtgacgcaa gagcgactcctgggggcggg 180 gcgaggcctt ggagatgcgt ccggaggcgg tggggagcga agactgacccggggccgtga 240 cgcggggcag gccctggggc gggggcgggt cctggggact ggggcgggcggccgaggccc 300 ggaagcggac tgaggcggcc tggagtctta gttggctact cgccatggacgaagcggatc 360 ggcggctcct gcggcggtgc cggctgcggc tggtggaaga gctgcaggtggaccagctct 420 gggacgtcct gctgagtcgc gagctgttca ggccccatat gatcgaggacatccagcggg 480 caggctctgg atctcggcgg gatcaggcca ggcagctgat catagatctggagactcgag 540 ggagtcaggc tcttcctttg ttcatctcct gcttagagga cacaggccaggacatgctgg 600 ctt 603 18 1137 DNA Homo sapiens CDS (22)...(822) 18agtcttagtt ggctactcgc c atg gac gaa gcg gat cgg cgg ctc ctg cgg 51 MetAsp Glu Ala Asp Arg Arg Leu Leu Arg 1 5 10 cgg tgc cgg ctg cgg ctg gtggaa gag ctg cag gtg gac cag ctc tgg 99 Arg Cys Arg Leu Arg Leu Val GluGlu Leu Gln Val Asp Gln Leu Trp 15 20 25 gac gcc ctg ctg agc cgc gag ctgttc agg ccc cat atg atc gag gac 147 Asp Ala Leu Leu Ser Arg Glu Leu PheArg Pro His Met Ile Glu Asp 30 35 40 atc cag cgg gca ggc tct gga tct cggcgg gat cag gcc agg cag ctg 195 Ile Gln Arg Ala Gly Ser Gly Ser Arg ArgAsp Gln Ala Arg Gln Leu 45 50 55 atc ata gat ctg gag act cga ggg agt caggct ctt cct ttg ttc atc 243 Ile Ile Asp Leu Glu Thr Arg Gly Ser Gln AlaLeu Pro Leu Phe Ile 60 65 70 tcc tgc tta gag gac aca ggc cag gac atg ctggct tcg ttt ctg cga 291 Ser Cys Leu Glu Asp Thr Gly Gln Asp Met Leu AlaSer Phe Leu Arg 75 80 85 90 act aac agg caa gca gca aag ttg tcg aag ccaacc cta gaa aac ctt 339 Thr Asn Arg Gln Ala Ala Lys Leu Ser Lys Pro ThrLeu Glu Asn Leu 95 100 105 acc cca gtg gtg ctc aga cca gag att cgc aaacca gag gtt ctc aga 387 Thr Pro Val Val Leu Arg Pro Glu Ile Arg Lys ProGlu Val Leu Arg 110 115 120 ccg gaa aca ccc aga cca gtg gac att ggt tctgga gga ttt ggt gat 435 Pro Glu Thr Pro Arg Pro Val Asp Ile Gly Ser GlyGly Phe Gly Asp 125 130 135 gtc gag cag aaa gac cat ggg ttt gag gtg gcctcc act tcc cct gaa 483 Val Glu Gln Lys Asp His Gly Phe Glu Val Ala SerThr Ser Pro Glu 140 145 150 gac gag tcc cct ggc agt aac ccc gag cca gatgcc acc ccg ttc cag 531 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp AlaThr Pro Phe Gln 155 160 165 170 gaa ggt ttg agg acc ttc gac cag ctg gacgcc ata tct agt ttg ccc 579 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp AlaIle Ser Ser Leu Pro 175 180 185 aca ccc agt gac atc ttt gtg tcc tac tctact ttc cca ggt ttt gtt 627 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser ThrPhe Pro Gly Phe Val 190 195 200 tcc tgg agg gac ccc aag agt ggc tcc tggtac gtt gag acc ctg gac 675 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp TyrVal Glu Thr Leu Asp 205 210 215 gac atc ttt gag cag tgg gct cac tct gaagac ctg cag tcc ctc ctg 723 Asp Ile Phe Glu Gln Trp Ala His Ser Glu AspLeu Gln Ser Leu Leu 220 225 230 ctt agg gtc gct aat gct gtt tcg gtg aaaggg att tat aaa cag atg 771 Leu Arg Val Ala Asn Ala Val Ser Val Lys GlyIle Tyr Lys Gln Met 235 240 245 250 cct ggt tgc ttt aat ttc ctc cgg aaaaaa ctt ttc ttt aaa aca tca 819 Pro Gly Cys Phe Asn Phe Leu Arg Lys LysLeu Phe Phe Lys Thr Ser 255 260 265 taa ggccagggcc cctcaccctg ccttatcttgcaccccaaag ctttcctgcc 872 ccaggcctga aagaggctga ggcctggact ttcctgcaactcaaggactt tgcagccggc 932 acagggtctg ctctttctct gccagtgaca gacaggctcttagcagcttc cagattgacg 992 acaagtgctg aacagtggag gaagagggac agatgaatgccgtggattgc acgtggcctc 1052 ttgagcagtg gctggtccag ggctagtgac ttgtgtcccatgatccctgt gttgtctcta 1112 gagcagggat taacctctgc actac 1137 19 908 DNAHomo sapiens 19 tccctctggg cacttgtctc ttgttgtgtc attgctgatt tgtctgattgcattccccat 60 tagaaatttg ctccttaagg actgagactg tccatggcag cattttcagcacttagcata 120 gtgctgggca tcttttgccc tgcaaaataa tgaacagatc agcaaaggctggtaaatggc 180 acacagtgca cctgcagctc cattgctgcc cacgtgcctg attgtttagacttacagtag 240 ctgggaaatg gggagacaag gtgagagcca tataaaagta cgtggataattagtatcctg 300 cctttctttc caaacagcgg gcaggctctg gatctcggcg ggatcaggccaggcagctga 360 tcatagatct ggagactcga gggagtcagg ctcttccttt gttcatctcctgcttagagg 420 acacaggcca ggacatgctg gcttcgtttc tgcgaactaa caggcaagcagcaaagttgt 480 cgaagccaac cctagaaaac cttaccccag tggtgctcag accagagattcgcaaaccag 540 aggttctcag accggaaaca cccagaccag tggacattgg ttctggaggatttggtgatg 600 tcggtaagta gcaagagagt gattggtggg tgggcatgac acgtagtcatttgggactca 660 ctatgagtac agctgggtgg gctgagattc agaatttaat tttagcattgtctttctgaa 720 tgttcatagg gatggcaaaa ttctgggagg ttttcaggag gttgaatcttggcagcccct 780 cctctgaaaa gcatgtgttg aggtctgttt tggatgtccc aggttttttgtttttgtttt 840 tgtttttttt cctgcctttg agatagcagg ccccaggttt tgatcaatgtttccagagcc 900 atctattg 908 20 731 DNA Homo sapiens unsure 536 unknown20 ctgtgtaatg ttctcttgtg tggctgtatc acaatttgta tatctaccaa actgttgtta 60gattggttgt ttccatgttg gggcgtgatg aataaggcct cagggaacat tttggccaag 120tcttttgtgc ccgtttgcac tccttgctct ttgatatata cctaagggtg gacttgctgt 180cagagggtag gtgtgtgttt aactttgtta gaaacctcta aacagatctc caaggtggct 240ctcccaccag cagagtctga gggtctgagt tctcgctgct ccgttcttgg ggggctgtcc 300tcacagagcc ccttgcccct cctctgctgt aacccaacat tcatgtttct ctctttctct 360gcctcttcag gtgctcttga gagtttgagg ggaaatgcag atttggtgag tctccatgaa 420atttgggcca gtagctcggg ctctgggccc ttccctcccc acccctcagg tccttccctg 480caggtgtgtc aggagcccag ggaacccaac cctaggcagt gcagtggggg tctggnctca 540gcctccctga ctccggccca gcgcctgctg gggcagattg ttcatctctg cgcttcctca 600tctgcaaagc tgacagcaga agcagggcag acctgatgag gggcagtcat cgggatccag 660accaagtcgc cactctctca ttcacaagta tttattgtgt ggctcccatg ctctgtgctg 720gggccacctg g 731 21 791 DNA Homo sapiens 21 tgccttagag aattacaaggggactgagcc tgggagaggg cagccttggg tgcctccagg 60 tggcctgggc atggttctgctgacagagtt ctcaggtaca ccccggccta ggtgacccgt 120 gaggtccttt gggatgaatgctcatggctg ttggatttcc aggttaccag ctcccttccc 180 ttaataccaa gatgggggggtcaccttcta gtgctagtct tccctgcagt tgcttctctt 240 ttgccctgga atgccgggtggagaccctgc tactccttgt gcgagagggc agaccccgtt 300 ttcttcccgc ctctccagactgtcctctag ggagcgtggg gagagcccgg gtttacgtgg 360 gtccctgtgc tggcaggcttacatcctgag catggagccc tgtggccact gcctcattat 420 caacaatgtg aacttctgccgtgagtccgg gctccgcacc cgcactggct ccaacatcga 480 ctgtgagaag ttgcggcgtcgcttctcctc gccgcatttc atggtggagg tgaagggcga 540 cctgactgcc aaggtacactcactatctgt ggagggagac agggtggggg gcagtgggtg 600 gggaagtatc ttttgagggactccaaaagc cagctgactc cccaggcgag cccttacatc 660 tggaggacca tgggaggtaggacggcccac ggctacaggt tcaagttcag acgcagaagt 720 ccgggtttga ggactggctctctcggccac ctgctgtgtg acctcctgtg cctcagtttc 780 cttgtctgga a 791 22 604DNA Homo sapiens 22 gggattacag gtgtgtgcca ccatgcctgg ctaaatttttttttttgtat ttttagtaga 60 gacggggttt caccatgttg gccaggctgg tctcgaactcgtgacctcaa atgatccacc 120 tgccttggcc tcccaaagtg ctgggattac aggcgtgaaccaccatgcct ggccaggctg 180 ggccctttta ttgttaggaa cggtccagtc tgcatctagacctatccgtg cttctggctc 240 acctgcagcc ctctcttgta gaaaatggtg ctggctttgctggagctggc gcagcaggac 300 cacggtgctc tggactgctg cgtggtggtc attctctctcacggctgtca ggtaggaagc 360 ctcccactgt tccctgggca ggcattgggt actggccgtgccaagaggct gtgcaggggc 420 catgtccctt cttgtgtcca aaacaccctt ggctttgtggaaaagggctg tggggccctg 480 cccacctcct gttgttttct tgggagccat gtggtcctctgaggagttgg ctgcaccgtc 540 ctgggcaggt cggtgttcct gggagaagcc ctctgggagagggagggcag agaccaggtc 600 tgct 604 23 810 DNA Homo sapiens 23 ccaagcgatattgatgccgc tgctgcaggg gccaaacttt gagaaccact ggtctagagg 60 ccgcagccagcactctccag gcacactggc ccctagaggc acatgagttt gacatgcaac 120 cccttgctgtcaaggtgtct ttccagatgc tgaccagggt ctttccagaa ccaacctgct 180 ctgtctcaatcagcctcgtg cccctggctt tgaccttcca aatccttggc tcccacaaaa 240 cctctgaacaggctgttgga ttcctactcc aacttcttgg ttttgtaacc agggttcttg 300 gtcttctaggccagccacct gcagttccca ggggctgtct acggcacaga tggatgccct 360 gtgtcggtcgagaagattgt gaacatcttc aatgggacca gctgccccag cctgggagga 420 aagcccaagctctttttcat ccaggcctgt ggtgggggta agcagctcct cagcctccct 480 ctgggtgggtctggtgggga gggagccgcc acctgcttct ttctccagcc tgcccctcac 540 agggcccatgaggtctctcc aggcagtcag agggtaccac acatggtccc tttttggcag 600 cacctctgtctggctggagc aggccctgct ctgtctgctt tgtagaggtc agagtcttct 660 gtttcatggcacagtggcat catgggctca agtaactttg gcagcttcaa ttccaaagac 720 ttggcaaaaagaaagcagaa gagagaggag aaacctcttt ttatttttct tgtcaccatt 780 ctcctgacctccatcaacta gaccccgttt 810 24 675 DNA Homo sapiens 24 tttatcagacaactatcttg gagctcacca ggaatttcct tgtgggcaaa atatgggaga 60 ggtgtgtagcttttcatctt gtagccatct tatttaggaa ccaaaacggg gaggcaggtt 120 tgcgtgacccagttcccagc ttgacttttc cgtttggctt aatgagtttg gggtcccaag 180 atttattttcctttcacact gttaatgtag tatattgcac cgactttcat atgttgaacc 240 atccttgtctgtgtatttca atgacaagag gtgtttgttt ctgcttcctg cagagcagaa 300 agaccatgggtttgaggtgg cctccacttc ccctgaagac gagtcccctg gcagtaaccc 360 cgagccagatgccaccccgt tccaggaagg tttgaggacc ttcgaccagc tggacgccat 420 atctagtttgcccacaccca gtgacatctt tgtgtcctac tctactttcc caggtgagca 480 catcagaagggctcgtcctc gcagccagtg ggtcttcccg tctgccctag aggcagctgt 540 gtggtgagagaaaagaccag ggtatgagtc ctggttctgt cttttgcccc tctgtgaccc 600 tgagcaagttacttctctct gagcctcggc tcaggctgag cgggcaagga tgcgcgctgc 660 aaggaggcctgcgcc 675 25 718 DNA Homo sapiens unsure 710 unknown 25 ggattacaggtgtgagccac tgtgcctggc agtttccttc ctttcttatg tcttggatga 60 gtcacttgacttttccaagc cttggtttcc ttgctgggaa aacaggaatt atagccggaa 120 tcaggggatggtcatgaggg tgaaataaga tcatgattag ggcagagcct caggggccag 180 gctgctcctgttggatgtat gtatgagtca aggtctggag ggcgcccgag ccagcgtgca 240 tggccagaggggtggtgggg agccggcgga ggggtggctc tccagcagtg ttcagccctc 300 ctccctccaaaggttttgtt tcctggaggg accccaagag tggctcctgg tacgttgaga 360 ccctggacgacatctttgag cagtgggctc actctgaaga cctgcagtcc ctcctgctta 420 gggtgagtgctgccttcctc tgcaaaggag aggggaggct gctgaggggc agcgtgtcct 480 cctggggctggggatttggg gtgagcaggg caggcccaaa ccaagggtaa aaggtagtag 540 actcctgcctctgagccttg gagtcggcac ttttgtgtct ctttatgaga ggcatcctgc 600 ctgtggtgacgtttgccctt tattcaaaga gttgccttcc ctgtctcctc caaggtcccc 660 aggtttgacacctcccgctc ctccactgat ggactggtcc tcagtgccan cgcagatg 718 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 gtagccaact aagactccag20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 gcttcgtccatggcgagtag 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28gagcctgccc gctggatgtc 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 ctggcctgat cccgccgaga 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 agctgcctgg cctgatcccg 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 tctatgatca gctgcctggc20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tgtcctctaagcaggagatg 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33ggcctgtgtc ctctaagcag 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 gccagcatgt cctggcctgt 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 ttgcctgtta gttcgcagaa 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 ctgagaacct ctggtttgcg20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 acatcaccaaatcctccaga 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38ctcaagagca ccgacatcac 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 gcatttcccc tcaaactctc 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 aggatgtaag ccaaatctgc 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 ctccatgctc aggatgtaag20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 atgaggcagtggccacaggg 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43cagaagttca cattgttgat 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 ccgcaacttc tcacagtcga 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 gcagtcaggt cgcccttcac 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 ttcttggcag tcaggtcgcc20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 cattttcttggcagtcaggt 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48gcaccatttt cttggcagtc 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 cagagcaccg tggtcctgct 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 accaccacgc agcagtccag 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 tgacagccgt gagagagaat20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 ggctggcctgacagccgtga 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53ggtggctggc ctgacagccg 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 gccgtagaca gcccctggga 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 tccatctgtg ccgtagacag 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 ggtcccattg aagatgttca20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 ggcctggatgaaaaagagct 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58cagagtgagc ccactgctca 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 agggactgca ggtcttcaga 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 cattagcgac cctaagcagg 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 ggcatctgtt tataaatccc20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gccctggccttatgatgttt 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63aagtccaggc ctcagcctct 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 aggaaagtcc aggcctcagc 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 ccggctgcaa agtccttgag 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 accctgtgcc ggctgcaaag20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 ggaagctgctaagagcctgt 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68cctccactgt tcagcacttg 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 ttcatctgtc cctcttcctc 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 ccacgtgcaa tccacggcat 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 gctcaagagg ccacgtgcaa20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 gatcatgggacacaagtcac 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73aggaagacgc gttactggca 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 aacctttttg tttggcacca 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 gtttgctgat ctatgagcga 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 ccggtttgct gatctatgag20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 catatttgcctcagatttat 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78ccatatttgc ctcagattta 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 ggacgcatct ccaaggcctc 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 gcctccggac gcatctccaa 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 ggtcagtctt cgctccccac20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 ccccgggtcagtcttcgctc 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83acggccccgg gtcagtcttc 20 84 20 DNA Artificial Sequence AntisenseOligonucleotide 84 ctttctgctc gacatcacca 20 85 20 DNA ArtificialSequence Antisense Oligonucleotide 85 ctatctcaaa ggcaggaaaa 20 86 20 DNAArtificial Sequence Antisense Oligonucleotide 86 tcaagagcac ctgaagaggc20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ggagactcaccaaatctgca 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88agagcccgag ctactggccc 20 89 20 DNA Artificial Sequence AntisenseOligonucleotide 89 acagggaccc acgtaaaccc 20 90 20 DNA ArtificialSequence Antisense Oligonucleotide 90 ggatgtaagc ctgccagcac 20 91 20 DNAArtificial Sequence Antisense Oligonucleotide 91 gtgagtgtac cttggcagtc20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 gccgagagagccagtcctca 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93gcaccatttt ctacaagaga 20 94 20 DNA Artificial Sequence AntisenseOligonucleotide 94 ggcttcctac ctgacagccg 20 95 20 DNA ArtificialSequence Antisense Oligonucleotide 95 ctgttcagag gttttgtggg 20 96 20 DNAArtificial Sequence Antisense Oligonucleotide 96 gagtaggaat ccaacagcct20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97 ggtggctggcctagaagacc 20 98 20 DNA Artificial Sequence Antisense Oligonucleotide 98ggagctgctt acccccacca 20 99 20 DNA Artificial Sequence AntisenseOligonucleotide 99 acctcttgtc attgaaatac 20 100 20 DNA ArtificialSequence Antisense Oligonucleotide 100 tctttctgct ctgcaggaag 20 101 20DNA Artificial Sequence Antisense Oligonucleotide 101 gaccttgactcatacataca 20 102 20 DNA Artificial Sequence Antisense Oligonucleotide102 aggaggacac gctgcccctc 20 103 20 DNA Artificial Sequence AntisenseOligonucleotide 103 gcactgagga ccagtccatc 20 104 1255 DNA Mus musculusCDS (1)...(1182) 104 atg gac gag gcg gac cgg cag ctc ctg cgg cga tgc agggtg cgc cta 48 Met Asp Glu Ala Asp Arg Gln Leu Leu Arg Arg Cys Arg ValArg Leu 1 5 10 15 gtg agc gag ctg caa gtc gcg gag ctc tgg gac gct ctgctg agt cga 96 Val Ser Glu Leu Gln Val Ala Glu Leu Trp Asp Ala Leu LeuSer Arg 20 25 30 gag ctc ttc acg cgc gac atg atc gag gat att cag cag gcagga tct 144 Glu Leu Phe Thr Arg Asp Met Ile Glu Asp Ile Gln Gln Ala GlySer 35 40 45 ggg tct cgg cgg gat cag gcc agg cag ctg gtc aca gac ctt gagacc 192 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Val Thr Asp Leu Glu Thr50 55 60 cga ggg agg cag gcc ctt cct ctc ttc atc tcc tgc tta gag gac aca240 Arg Gly Arg Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr 6570 75 80 ggc caa ggc acc ctg gct tca ctc ttg caa agc ggt cgg caa gca gcc288 Gly Gln Gly Thr Leu Ala Ser Leu Leu Gln Ser Gly Arg Gln Ala Ala 8590 95 aag cag gat cca gag gct gtt aaa ccc cta gac cac ctg gtg cct gtg336 Lys Gln Asp Pro Glu Ala Val Lys Pro Leu Asp His Leu Val Pro Val 100105 110 gtc ctg gga cca atg gga ctc aca gca aag gag cag aga gta gtg aag384 Val Leu Gly Pro Met Gly Leu Thr Ala Lys Glu Gln Arg Val Val Lys 115120 125 ctg gag ccg tca cag cct gcc gtg gga aac ctc acc cca gtg gtg ctg432 Leu Glu Pro Ser Gln Pro Ala Val Gly Asn Leu Thr Pro Val Val Leu 130135 140 ggg cca gaa gag ctc tgg cct gct cgg ctc aag cca gag gtt ctc aga480 Gly Pro Glu Glu Leu Trp Pro Ala Arg Leu Lys Pro Glu Val Leu Arg 145150 155 160 cca gaa aca ccc agg ccg gtg gac att ggt tct ggc gga gct catgat 528 Pro Glu Thr Pro Arg Pro Val Asp Ile Gly Ser Gly Gly Ala His Asp165 170 175 gtc tgt gtt cca ggg aag atc agg gga cat gca gat atg gca tacacc 576 Val Cys Val Pro Gly Lys Ile Arg Gly His Ala Asp Met Ala Tyr Thr180 185 190 ctg gat tcg gat ccc tgt ggc cac tgc ctc atc atc aac aat gtgaac 624 Leu Asp Ser Asp Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn195 200 205 ttc tgc cct tcc tcg ggg ctc ggc aca cgc acg ggc tcc aac ttggac 672 Phe Cys Pro Ser Ser Gly Leu Gly Thr Arg Thr Gly Ser Asn Leu Asp210 215 220 cgt gac aaa ctt gag cac cga ttc cgc tgg ctg cgc ttc atg gtggag 720 Arg Asp Lys Leu Glu His Arg Phe Arg Trp Leu Arg Phe Met Val Glu225 230 235 240 gtg aag aac gac ctg act gcc aag aaa atg gtc acg gct ttgatg gag 768 Val Lys Asn Asp Leu Thr Ala Lys Lys Met Val Thr Ala Leu MetGlu 245 250 255 atg gca cac cgg aac cac cgt gcc ctg gac tgc ttt gtg gtggtc atc 816 Met Ala His Arg Asn His Arg Ala Leu Asp Cys Phe Val Val ValIle 260 265 270 ctc tct cat ggc tgc cag gcc agc cac ctc cag ttc ccg ggtgct gtc 864 Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly AlaVal 275 280 285 tat ggg aca gat gga tgc tcc gtg tcc att gag aaa att gtgaat atc 912 Tyr Gly Thr Asp Gly Cys Ser Val Ser Ile Glu Lys Ile Val AsnIle 290 295 300 ttc aac ggg agc ggc tgc ccc agc ctg gga ggg aag ccc aagctc ttc 960 Phe Asn Gly Ser Gly Cys Pro Ser Leu Gly Gly Lys Pro Lys LeuPhe 305 310 315 320 ttc atc cag gcc tgc ggt ggt gag cag aaa gac cat ggcttt gag gtg 1008 Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly PheGlu Val 325 330 335 gcc tgc act tcc tct caa ggc agg acc ttg gac agt gactct gag cca 1056 Ala Cys Thr Ser Ser Gln Gly Arg Thr Leu Asp Ser Asp SerGlu Pro 340 345 350 gat gct gtc cca tat cag gaa ggc cca agg ccc ttg gaccag ctg gat 1104 Asp Ala Val Pro Tyr Gln Glu Gly Pro Arg Pro Leu Asp GlnLeu Asp 355 360 365 gct gtg tca agt ttg cct acc ccc agt gac atc ctt gtgtcc tac tcc 1152 Ala Val Ser Ser Leu Pro Thr Pro Ser Asp Ile Leu Val SerTyr Ser 370 375 380 acc ttc cca ggt cgc caa tgc tgt ttc tga gaaagggacttacaagcaga 1202 Thr Phe Pro Gly Arg Gln Cys Cys Phe 385 390 ttcctggctgttttaacttc ctccggaaaa agctgttttt taaaacttca tga 1255 105 20 DNAArtificial Sequence Antisense Oligonucleotide 105 accctgcatc gccgcaggag20 106 20 DNA Artificial Sequence Antisense Oligonucleotide 106gcaccctgca tcgccgcagg 20 107 20 DNA Artificial Sequence AntisenseOligonucleotide 107 cactaggcgc accctgcatc 20 108 20 DNA ArtificialSequence Antisense Oligonucleotide 108 tgaatatcct cgatcatgtc 20 109 20DNA Artificial Sequence Antisense Oligonucleotide 109 ctgaatatcctcgatcatgt 20 110 20 DNA Artificial Sequence Antisense Oligonucleotide110 cctgctgaat atcctcgatc 20 111 20 DNA Artificial Sequence AntisenseOligonucleotide 111 cgccgagacc cagatcctgc 20 112 20 DNA ArtificialSequence Antisense Oligonucleotide 112 caaggtctgt gaccagctgc 20 113 20DNA Artificial Sequence Antisense Oligonucleotide 113 tctcaaggtctgtgaccagc 20 114 20 DNA Artificial Sequence Antisense Oligonucleotide114 ctccctcggg tctcaaggtc 20 115 20 DNA Artificial Sequence AntisenseOligonucleotide 115 gaagagagga agggcctgcc 20 116 20 DNA ArtificialSequence Antisense Oligonucleotide 116 aagccagggt gccttggcct 20 117 20DNA Artificial Sequence Antisense Oligonucleotide 117 ttgcaagagtgaagccaggg 20 118 20 DNA Artificial Sequence Antisense Oligonucleotide118 tgccgaccgc tttgcaagag 20 119 20 DNA Artificial Sequence AntisenseOligonucleotide 119 aggcaccagg tggtctaggg 20 120 20 DNA ArtificialSequence Antisense Oligonucleotide 120 ctcctttgct gtgagtccca 20 121 20DNA Artificial Sequence Antisense Oligonucleotide 121 ctgctcctttgctgtgagtc 20 122 20 DNA Artificial Sequence Antisense Oligonucleotide122 acggctccag cttcactact 20 123 20 DNA Artificial Sequence AntisenseOligonucleotide 123 tgtgacggct ccagcttcac 20 124 20 DNA ArtificialSequence Antisense Oligonucleotide 124 aggctgtgac ggctccagct 20 125 20DNA Artificial Sequence Antisense Oligonucleotide 125 acggcaggctgtgacggctc 20 126 20 DNA Artificial Sequence Antisense Oligonucleotide126 gtttcccacg gcaggctgtg 20 127 20 DNA Artificial Sequence AntisenseOligonucleotide 127 tgagaacctc tggcttgagc 20 128 20 DNA ArtificialSequence Antisense Oligonucleotide 128 tctgagaacc tctggcttga 20 129 20DNA Artificial Sequence Antisense Oligonucleotide 129 tggtctgagaacctctggct 20 130 20 DNA Artificial Sequence Antisense Oligonucleotide130 ccaatgtcca ccggcctggg 20 131 20 DNA Artificial Sequence AntisenseOligonucleotide 131 ccctgatctt ccctggaaca 20 132 20 DNA ArtificialSequence Antisense Oligonucleotide 132 ccatatctgc atgtcccctg 20 133 20DNA Artificial Sequence Antisense Oligonucleotide 133 cagggtgtatgccatatctg 20 134 20 DNA Artificial Sequence Antisense Oligonucleotide134 aatccagggt gtatgccata 20 135 20 DNA Artificial Sequence AntisenseOligonucleotide 135 cgaatccagg gtgtatgcca 20 136 20 DNA ArtificialSequence Antisense Oligonucleotide 136 attgttgatg atgaggcagt 20 137 20DNA Artificial Sequence Antisense Oligonucleotide 137 acattgttgatgatgaggca 20 138 20 DNA Artificial Sequence Antisense Oligonucleotide138 gtcacggtcc aagttggagc 20 139 20 DNA Artificial Sequence AntisenseOligonucleotide 139 tgctcaagtt tgtcacggtc 20 140 20 DNA ArtificialSequence Antisense Oligonucleotide 140 agcggaatcg gtgctcaagt 20 141 20DNA Artificial Sequence Antisense Oligonucleotide 141 cttcacctccaccatgaagc 20 142 20 DNA Artificial Sequence Antisense Oligonucleotide142 tcttcacctc caccatgaag 20 143 20 DNA Artificial Sequence AntisenseOligonucleotide 143 tcttggcagt caggtcgttc 20 144 20 DNA ArtificialSequence Antisense Oligonucleotide 144 gcacggtggt tccggtgtgc 20 145 20DNA Artificial Sequence Antisense Oligonucleotide 145 agcagtccagggcacggtgg 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide146 ccacaaagca gtccagggca 20 147 20 DNA Artificial Sequence AntisenseOligonucleotide 147 gaggatgacc accacaaagc 20 148 20 DNA ArtificialSequence Antisense Oligonucleotide 148 gcctggcagc catgagagag 20 149 20DNA Artificial Sequence Antisense Oligonucleotide 149 gtggctggcctggcagccat 20 150 20 DNA Artificial Sequence Antisense Oligonucleotide150 gacagcaccc gggaactgga 20 151 20 DNA Artificial Sequence AntisenseOligonucleotide 151 attcacaatt ttctcaatgg 20 152 20 DNA ArtificialSequence Antisense Oligonucleotide 152 caaagccatg gtctttctgc 20 153 20DNA Artificial Sequence Antisense Oligonucleotide 153 aggccacctcaaagccatgg 20 154 20 DNA Artificial Sequence Antisense Oligonucleotide154 gtcctgcctt gagaggaagt 20 155 20 DNA Artificial Sequence AntisenseOligonucleotide 155 atctggctca gagtcactgt 20 156 20 DNA ArtificialSequence Antisense Oligonucleotide 156 gcatccagct ggtccaaggg 20 157 20DNA Artificial Sequence Antisense Oligonucleotide 157 ttgacacagcatccagctgg 20 158 20 DNA Artificial Sequence Antisense Oligonucleotide158 taggcaaact tgacacagca 20 159 20 DNA Artificial Sequence AntisenseOligonucleotide 159 ggtaggcaaa cttgacacag 20 160 20 DNA ArtificialSequence Antisense Oligonucleotide 160 aggtggagta ggacacaagg 20 161 20DNA Artificial Sequence Antisense Oligonucleotide 161 aacctgggaaggtggagtag 20 162 20 DNA Artificial Sequence Antisense Oligonucleotide162 tttcttgtcc ctccaggaga 20 163 20 DNA Artificial Sequence AntisenseOligonucleotide 163 gagccacttt tcttgtccct 20 164 20 DNA ArtificialSequence Antisense Oligonucleotide 164 taccaggagc cacttttctt 20 165 20DNA Artificial Sequence Antisense Oligonucleotide 165 gatgtaccaggagccacttt 20 166 20 DNA Artificial Sequence Antisense Oligonucleotide166 tccaaggtct cgatgtacca 20 167 20 DNA Artificial Sequence AntisenseOligonucleotide 167 agaatgccat ccaaggtctc 20 168 20 DNA ArtificialSequence Antisense Oligonucleotide 168 actgctccag aatgccatcc 20 169 20DNA Artificial Sequence Antisense Oligonucleotide 169 accctgagaaggagggactg 20 170 20 DNA Artificial Sequence Antisense Oligonucleotide170 aagtcccttt ctcagaaaca 20 171 20 DNA Artificial Sequence AntisenseOligonucleotide 171 cagccaggaa tctgcttgta 20 172 20 DNA ArtificialSequence Antisense Oligonucleotide 172 ttttccggag gaagttaaaa 20 173 20DNA Artificial Sequence Antisense Oligonucleotide 173 cagctttttccggaggaagt 20 174 20 DNA Artificial Sequence Antisense Oligonucleotide174 gcattggcga cctgggaagg 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targetedto a nucleic acid molecule encoding caspase 9, wherein said compoundspecifically hybridizes with and inhibits the expression of caspase 9.2. The compound of claim 1 which is an antisense oligonucleotide.
 3. Thecompound of claim 2 wherein the antisense oligonucleotide has a sequencecomprising SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76,77, 78, 80, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 100, 101, 102, 103, 105, 106, 108, 109, 110, 111, 112, 113, 114,116, 117, 118, 122, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134,135, 136, 137, 139, 140, 141, 143, 144, 145, 146, 147, 150, 152, 154,155, 156, 157, 158, 159, 161, 162, 163, 164, 165, 166, 168, 169, 170,171, 172, 173 or
 174. 4. The compound of claim 2 wherein the antisenseoligonucleotide comprises at least one modified internucleoside linkage.5. The compound of claim 4 wherein the modified internucleoside linkageis a phosphorothioate linkage.
 6. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified sugar moiety.7. The compound of claim 6 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified nucleobase. 9.The compound of claim 8 wherein the modified nucleobase is a5-methylcytosine.
 10. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50nucleobases in length which specifically hybridizes with at least an8-nucleobase portion of an active site on a nucleic acid moleculeencoding caspase
 9. 12. A composition comprising the compound of claim 1and a pharmaceutically acceptable carrier or diluent.
 13. Thecomposition of claim 12 further comprising a colloidal dispersionsystem.
 14. The composition of claim 12 wherein the compound is anantisense oligonucleotide.
 15. A method of inhibiting the expression ofcaspase 9 in cells or tissues comprising contacting said cells ortissues with the compound of claim 1 so that expression of caspase 9 isinhibited.
 16. A method of treating an animal having a disease orcondition associated with caspase 9 comprising administering to saidanimal a therapeutically or prophylactically effective amount of thecompound of claim 1 so that expression of caspase 9 is inhibited. 17.The method of claim 16 wherein the disease or condition is stroke, braininjury or a neurodegenerative disease.
 18. The method of claim 16wherein the disease or condition is a hyperproliferative disorder. 19.The method of claim 16 wherein the disease or condition is ahaematopoetic disorder.
 20. The method of claim 16 wherein the diseaseor condition is a bone metabolism or cholesterol disorder.